the pathophysiological role of neutrophil extracellular ...€¦ · the pathophysiological role of...

The Pathophysiological Role of NeutrophilExtracellular Traps in Inflammatory DiseasesAldo Bonaventura1,� Luca Liberale1,2,� Federico Carbone1 Alessandra Vecchié1

Candela Diaz-Cañestro2 Giovanni G. Camici2 Fabrizio Montecucco1,3,4 Franco Dallegri1,3

1Department of Internal Medicine, First Clinic of Internal Medicine,University of Genoa, Genoa, Italy

2Centre for Molecular Cardiology, University of Zürich,Schlieren, Switzerland

3Ospedale Policlinico San Martino, Genoa, Italy4Centre of Excellence for Biomedical Research (CEBR), University ofGenoa, Genoa, Italy

Thromb Haemost 2018;118:6–27.

Address for correspondence Aldo Bonaventura, MD, Department ofInternal Medicine, University of Genoa, 6 viale Benedetto XV, 16132Genoa, Italy (e-mail: [email protected]).

Introduction

Neutrophil extracellular traps (NETs)havebeenrecently recog-nized as a part of the wide defence strategy of neutrophils. In2004, Brinkmann et al found that the induction of NETs by

interleukin (IL)-8 and lipopolysaccharide (LPS) supported theirformationduring inflammation andbacterial infection, as seenin the course of appendicitis.1 From this pivotal work, neutro-phil alternative death pathway, named NETosis,2 has beenindicated as a further way to act in innate immune defenceeven after their death. In recent years, NETs have been sug-gested to play a role also in non-infectious diseases, such as

Keywords

► NETs► infectious diseases► autoimmune diseases► metabolic disorders► cancer

Abstract Neutrophil pathogen-killing mechanism termed neutrophil extracellular traps (NETs)has been recently identified. NETs consist of chromatin and histones along with serineproteases and myeloperoxidase and are induced by a great variety of infectious andnon-infectious stimuli. NETosis is a kind of programmed neutrophil death characterizedby chromatin decondensation and release of nuclear granular contents, mainly drivenby peptidylarginine deiminase 4 citrullination of histones. Although classically relatedto the protection against infectious pathogens, nowadays NETs have been described asa player of several pathophysiological processes. Neutrophil dysregulation has beendemonstrated in the pathogenesis of most representative vascular diseases, such asacute coronary syndrome, stroke and venous thrombosis. Indeed, NETs have beenidentified within atherosclerotic lesions and arterial thrombi in both human beings andanimal models. Moreover, an imbalance in this mechanism has been proposed as acritical source of modified and/or externalized autoantigens in autoimmune andinflammatory diseases. Finally, an update on the role of NETs in the pathogenesis ofcancer has been included. In the present review, based on papers released on PubMedand MEDLINE up to July 2017, we point to update the knowledge on NETs, from theirstructure to their roles in infectious diseases as well as in cardiovascular diseases,autoimmunity, metabolic disorders and cancer, with a look to future perspectives andtherapeutic opportunities.

� These authors equally contributed to this work as first authors.

receivedSeptember 13, 2017accepted after revisionOctober 22, 2017

Copyright © 2018 Schattauer DOI https://doi.org/10.1160/TH17-09-0630.ISSN 0340-6245.

Review Article6

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mailto:[email protected]

https://doi.org/10.1160/TH17-09-0630

https://doi.org/10.1160/TH17-09-0630

systemic lupuserythematosus (SLE), rheumatoidarthritis (RA),vasculitis, diabetes, atherosclerosis and cancer.

In the present review, based on papers released onPubMed and MEDLINE up to July 2017 (searched terms incombination: NETs, neutrophils, infections, autoimmunity,cancer, diabetes, cardiovascular [CV] diseases; articles havealso been retrieved through searches of reference lists andauthors’ files), we aimed at updating knowledge on NETs,from their structure to their roles in infectious diseases aswell as in CV disease, autoimmunity, metabolic disorders,and cancer, finally concentrating on future perspectives interms of clinical usefulness for therapeutic purposes.

Structure and Formation of NETs

NETs are constituted by extracellular strands of unwoundDNA usually in complex with histones and proteins fromneutrophil primary, secondary and tertiary granules, includ-ing componentswith inflammatory and bactericidal activity,such as neutrophil elastase (NE), myeloperoxidase (MPO),cathepsin G, lactoferrin, pentraxin 3, gelatinase, proteinase 3and peptidoglycan-binding proteins. Generally, most of theneutrophil DNA is transcriptionally inactive and condensedinto heterochromatinwithin the nucleus, with DNAwrappedaround histones to form nucleosomes. Chromatin deconden-sation begins NETosis and is mediated by peptidyl arginasedeaminase 4 (PAD4) catalyzing the conversion of histonearginines to citrullines, which weakens histone–DNA bind-ing and consequently unwraps nucleosomes. A central role in

NET regulation is played by intracellular calcium as a secondmessenger of neutrophil activation,3 particularly PAD4 isactivated by calcium itself.4 NE is essential for NET produc-tion, too, as it cleaves histones during NET formation(►Fig. 1).5

Starting from in vitro studies, distinct activation pathwaysfor NET formation have been identified and are believed to beactive in vivo as well. These pathways include activation byintegrins and toll-like receptors (TLRs)6,7 as signals triggeringNETosis in response to bacterial infections.8 In this setting, L-selectin-mediated signals have also been described to elicitNETs in vitro.8Most in vitro studies to identify mechanisms offormation of NETs used phorbol 12-myristate 13-acetate(PMA), despite this being an artificial trigger bypassing mem-brane receptors and their specific intracellular pathways.Actually, the widely described critical role of nicotinamideadenine dinucleotide phosphate oxidase (NOX) and MPO forNET formationmay be linked to the in vitro activation by PMA.Inchronicgranulomatousdisease (CGD)patients, a critical roleof NOX in NET formation was confirmed in vivo.9

To date, three different models of NET formation havebeen identified. The best described one is called “suicidalNETosis” and lasts from 2 to 4 hours.10 After neutrophilactivation, NOX increases its activity via the protein kinase C(PKC)/rapidly accelerated fibrosarcoma (Raf)/mitogen-acti-vated protein kinase ERK kinase (MERK)/extracellular signal-regulated kinase (ERK) complex leading to cytosolic calciumintake, PAD4 activation and chromatin decondensation. Oncethe increase in cytosolic calcium takes place, PAD4 activation

Fig. 1 Viable neutrophil extracellular trap (NET) formation. After neutrophil activation, peptidyl arginase deaminase 4 (PAD4) citrullinates somehistone arginines to weaken the tight electrostatic binding between histones and DNA in nucleosomes. The immediate consequence is that bothnuclear and granule membranes dissolved. At this moment, DNA is decondensed and meets citrullinated histones and granule proteins, whichare all expelled from neutrophils as NETs ready to catch and even kill microbes. The last step sees the surface membrane coming intact leaving aviable neutrophil without nucleus.

Thrombosis and Haemostasis Vol. 118 No. 1/2018

NETs in Infectious and Non-infectious Diseases Bonaventura et al. 7

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and chromatin decondensation occur.11 Then, ROS acts assecond messengers by promoting the loss of the nuclearmembrane. In this way, chromatin spreads throughout thecytoplasm mixing with cytoplasmic proteins and granulemediators and is finally released outside the cell throughmembrane pores and cellular lysis. Differently, during vitalNET generation, neutrophils release NETs with no loss ofnuclear or plasma membrane within 5 to 60 minutes inde-pendently of ROS and Raf/MERK/ERK pathway. This processevolves through different stages characterized by morpho-logical changes: (1) nuclear envelope growth and vesiclerelease, (2) nuclear decondensation and (3) nuclear envelopedisruption.12 Typically, this type of NET production is sti-mulated by the recognition of stimuli via TLRs and the C3complement receptor6,13 as well as via the interactionbetween glycoprotein Ib in platelets with β2 integrin inneutrophils by the activation of ERK.14 Finally, anothertype of vital, ROS-dependent NET generation has beendescribed, in which mitochondrial DNA is released insteadof nuclear DNAwith NET formationwithin 15minutes by therecognition of C5a or LPS.15

By definition, neutrophils are widely recognized as theonly producers of NETs, although only a limited part of them(around 20% depending on the stimulus) owns this skillcontributing to the rising concept of neutrophils being aheterogeneous class of cells.16 Anyway, it is not knownwhether these phenotypic differences could be relevant tothe ability of these neutrophils to undergo NETosis. Actually,the formation of extracellular traps (ETs) is far known to berestricted to cells of myeloid origin, even if the classicalrequirements for NET formation in PMA-stimulated neutro-phils—such as respiratory burst, NE and MPO—are mainlymet by neutrophils. However, eosinophils have also beendescribed to generate a respiratory burst and their perox-idase can convert hydrogen peroxide into oxidizing halogenderivatives.17 These eosinophil ETs contain intact eosinophilgranules, can catch bacteria and have been demonstratedin vivo in eosinophil-rich secretions.18,19 Basophils, too, canform ETs killing bacteria, but independently from NOXactivity.20,21 On the contrary, mast cells were shown torelease NOX-dependent ETs in vitro when stimulated byeither Staphylococcus pyogenes or PMA.22 Inmice,monocytesand macrophages can form ETs when stimulated by PMA.23

NETs and Infections

NETs have been demonstrated to own a broad effectivenessagainst different pathogens, such as bacteria, viruses, fungi,and parasites. Anyway, experimental data indicate that NETstrigger is restricted to specific microorganisms. To date, theprecise role of NETs in sepsis has not been completelyelucidated. NETs have been suggested to reduce bacterialspreading, especially in the early phase of infection,24 buttheir role appears to be limited: neither the lack of PAD4 northe treatment with deoxyribonuclease (DNAse) impacted onbacterial load in animals subjected to sepsis.25,26 Further-more, the excessive formation of NETs during sepsis isassociated with organ damage. Interactions among platelets,

neutrophils and activated endothelial cells lead to anincreased formation of NETs, which interact with vascularendothelium ultimately leading to endothelial damage andorgan injury in a histone- and MPO-dependent manner.27

Besides, histones can stimulate TLR2 and TLR4 to enhance theproduction of proinflammatory mediators via MyD88 sig-naling.28,29Considering all thesefindings, it is clear that NETsmight have a detrimental role in sepsis. Accordingly, bothantibiotic therapy and anti-NETosis conditions, such asDNAse treatment or abrogation of PAD4, havebeen describedto increase the survival rate of animals26,30,31 and humans32

with sepsis by reducing NET burden. Microorganisms indu-cing NET production are indicated in ►Table 1.

BacteriaA great number of Gram-positive and Gram-negative bacteriahave been demonstrated to trigger NET formation. Brinkmannet al have used Staphylococcus aureus in 2004 in their seminalwork as a stimulus to investigate NETosis.1 Some years later,Pilsczek et al deepened the previous finding by describing afaster, ROS-independent NET production in response to Sta-phylococcus-related infection, named vital NET release.10

S. aureus carries several virulence factors, among which leu-kotoxinGHandPanton–Valentine leukocidin canpromoteNETformation via an oxidative mechanism.33 Despite this, thebacterium has evolved different mechanisms to escape NETkilling. For example, S. aureus can express pore-formingvirulence factorsneutralizingneutrophilsby inducingnecrosisat the expense of NETosis.34 Besides, its catalase expressionblocks the stack of hydrogen peroxide, thus protecting thebacterium from intracellular oxidation and NETs.35 Further-more,methicillin-resistant S. aureus (MRSA) has been found toexpress extracellular nucleases for biofilm dispersal anddegradation of NETs. As a proof of this, mice infected byMRSA presented with a higher mortality with respect tocontrols infected with a nuclease-deficient strain that ismore susceptible to extracellular killing by neutrophils.36

Streptococcus pneumoniae and S. pyogenes can induceNETosis, but they also developed some escape mechanisms.S. pyogenes virulence factorM1modulates NET formation viaan association with fibrinogen ultimately leading to a com-plex, which stimulates neutrophils.37 Furthermore, overex-pression of M1 in susceptible strains of S. pyogenes confersresistance to extracellular killing because of the sequestra-tion and neutralization of the LL37 (also known as cathe-licidin), which is a neutrophil antimicrobial peptidesignificantly decreasing bacterial colonization.38 Mutantforms of M1 have been found with a decreased ability ofNET induction and deletion of M1 increases the tendencytoward NET killing.7 Similarly, α-enolase from S. pneumoniaecan increase neutrophil-migrating activity and induce theirdeath by releasing NETs; however, genetic ablation of α-enolase has not blocked NETosis.39 S. pneumoniae can escapeNETs in a passive manner through its polysaccharide capsulereducing NET binding40 or trough active strategies. S. pneu-moniae can express the DNase EndA, which facilitates theescape from NETs increasing the virulence in vivo.41 In asimilar way, the nuclease Sda1 in S. pyogenes degrades NETs


NETs in Infectious and Non-infectious Diseases Bonaventura et al.8

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and confers high virulence in vivo.42 Some strains of Strep-tococci can express the protease SpeB, which degrades Sda1blocking the possibility to escape NETs, as found in mousemodels.43 Interestingly, Sda1 has been demonstrated todegrade bacterial DNA, thus preventing the alert of theimmune system via TLR9.44 This witnesses the virulenceattributed to Sda1 which does not depend merely on NETescape, but probably on an intrinsic capacity.

NETs have been described to be significantly inducedwhenneutrophils are stimulated with the serum of patients suffer-ing from septic shock by Escherichia coli and this is likely todepend on TLR or complement receptor activation.45 Theenteropathogenic strain WS2572 of E. coli can trigger NETformation in neutrophils from the bone marrow of wild-typemice. Interestingly, NET synthesis is abolished in neutrophilsfrom glutathione reductase (GSR)-deficient mice suggesting a

Table 1 Microorganisms inducing NETosis

Authors Microorganism Microbe peptidesinducing NETs

Effects Type of NETosis

Bacteria

Pilsczek et al10 S. aureus Leukotoxin GH;Panton-Valentineleukocidin

ROS-independent inductionwith nuclear DNA liberation,TLR2- and C3-dependent

Vital

Mori et al39 S. pneumoniae EndA; α-enolase ROS-independent induction Suicidal

Carestia et al,14

Marin-Esteban et al,226

Pieterse et al227

E. coli Unknown Platelet-free or platelet-de-pendent induction, TLR4-de-pendent or independent, andROS-dependent orindependent

Suicidal (in absence ofplatelets) or vital (in presenceof platelets)

Brinkmann et al1 S. flexneri IcsA and IpaB Induction Suicidal

Brinkmann et al1 S. typhimurium Unknown Induction Suicidal

Möllerherm et al,49

Gillenius and Urban228Yersinia spp. Yops proteins and

invasion proteinROS-dependent induction,PI3K signaling, β-integrinpathway

Suicidal

Braian et al51 M. tuberculosis Adhesins, ESAT/6;hsp72 is releasedafter M. tuberculosisphagocytosis

Phagocytosis-, elastase, andROS-dependent induction

Suicidal

Seper et al17 V. cholerae Dns and Xds ROS-dependent induction Suicidal

Viruses

Tripathi et al54 Influenza virus Unknown ROS- and PAD4-dependentinduction

Suicidal

Moreno-Altamirano et al55 Dengue virus Unknown NET inhibition and Glut-1decreasing glucose capture

Unknown

Saitoh et al52 HIV Unknown ROS-dependent induction Suicidal

Raftery et al57 Hantavirus Viral particles,Src kinase, andβ2 integrin

ROS-dependent induction Suicidal

Fungi

Byrd et al229 C. albicans Unknown C3R- and fibronectin-depen-dent and ROS-independentinduction

Vital

Bruns et al60 A. fumigatus RodA ROS-dependent induction;spores containing RodA donot induce NETs

Suicidal

Rocha et al61 C. neoformans Unknown ROS and NETs inhibition Unknown

Parasites

Waisberg et al230 P. falciparum Agaphelin Induction through P. falciparumand inhibition throughagaphelin

Suicidal

Abi Abdallah et al65 T. gondii Unknown MEK-ERK-dependentinduction

Suicidal

Abbreviations: C3R, C3 receptor; ESAT, early secretory antigen target; HIV, human immunodeficiency virus; hsp, heat shock protein; MEK-ERK,mitogen-activated protein kinase/extracellular signal regulated kinase; NET, neutrophil extracellular trap; PAD, protein arginine deiminase; PI3K,phosphatidylinositol 3-kinase; ROS, reactive oxygen species; TLR, toll-like receptor.



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role for oxidative levels in the formation of NETs.46 As LL37 isassociated with NET synthesis, it may play a relevant role inpathogen elimination by cooperating with NETs.47

Clostridium difficile is likely to stimulate NET formation,which may act to reach the injured areas of the intestinalepithelium and effectively hinder bacterial dissemination.48

Brinkmann et al demonstrated that Shigella flexneri is trappedby NETs in vitro and described the ability of DNA-associatedelastase to abolish virulence factors IcsA and IpaB.1 Salmonellatyphimurium has been shown to trigger NETs. S. typhimuriumis usually trapped and eliminated by components of NETs,including granular proteins and H2A histone.1 In 2015, Möl-lerherm et al demonstrated that some serotypes of Yersiniaenterocolitica could induce NETs in vitro within 1 hour ofincubation, but induction diminished as the incubation timeincreased, maybe related to the effects of calcium- and mag-nesium-dependent nucleases.49 Vibrio cholerae can induceNETs in vitro after the contact with neutrophils. However, itis able to release the nucleases Dns and Xds as an evasionmechanism, thus feeding the infectious process.17 Finally, thefacultative intracellular bacterium Mycobacterium tuberculo-sis has also been demonstrated to induce NETs when co-cultured with neutrophils. Although NETs effectively trapand hinder the spread ofM. tuberculosis, NET-derived compo-nents do not kill them.50 Braian et al proposed a role for theheat shock protein 72 sequestered in NETs in the interactionbetweenneutrophils andmacrophagesduring the early innateimmune phase of an infection by M. tuberculosis.51

VirusesNeutrophils are slightly involved in viral infections and onlyfew studies investigated this issue. After neutrophils recog-nize human immunodeficiency virus (HIV)-1 nucleic acidsthrough TLR7 and TLR8, they release ROS inducing NETs,which capture and neutralize HIV virions by MPO and α-defensins.52 At the same time, HIV-1 is also able to suppressNET formation being recognized by CD209 on dendritic cells(DCs) and leading them to the production of anti-inflamma-tory IL-10.52 Influenza A virus stimulates NETs via PAD4,while NET-associated α-defensin-1 blocks its replication byabrogating protein kinase C pathway.53 Also LL37 is involvedin NET production in response to influenza A virus in vitro,while arginine-rich H3 and H4 histones are important forviral aggregation and neutralization.54 A role for NETs hasalso been demonstrated for dengue virus55 and respiratorysyncytial virus, although for the latter NETs can contribute toairway obstruction, thus exerting a dual protective andpathogenic role.56 NETs have also been detected in kidneybiopsies and sera of patients infected by hantavirus.57 Han-taan virus (HTNV), the prototype member of the genusHantavirus, uses the β2 integrin complement receptor (CR)3 and CR4 as entry receptors, but can concurrently induceboth ROS production and NET formation through the sameβ2 integrin signaling. This systemic NET overflow is accom-panied by the production of autoantibodies towards nuclearantigens. Moreover, HTNV has been found to stimulatehuman NETs in a more efficient way and at lower titrescompared with vaccinia virus or LPS.57

FungiNeutrophils play a crucial role in the control of fungalinfections, with NETs behaving as a precious weapon forthis purpose. Candida albicans can change from yeast tohyphae; since hyphae are too big to be phagocytosed, extra-cellular killing by NETs is a perfect strategy to block this formas well as an efficient way to kill C. albicans as a single cell.58

Calprotectin has been identified as a major antifungal agenttoward C. albicans and in fact is found to be associatedwith itin NETs; anyway, direct contact with the fungus is notrequired as calprotectin can chelate magnesium and zincions requested for Candida growth.59

Aspergillus fumigatus induces NET release in vitro requir-ing NOX.35 Although NETs are fundamental for the elimina-tion of A. fumigatus hyphae, they are not induced by sporesbecause of the presence of RodA in the wall of spore cells.60

RodA-deficient A. fumigatus conidia induce NETs betterthan wild-type hyphae, so that RodA may have an inhibitoryeffect on NET formation by shielding yet unidentified NET-inducing elements in conidia. As a further proof of it, A.fumigatus conidia are killed primarily by phagocytosis andnot by NETs.60

Cryptococcus neoformans can modulate NET production.Particularly, neutrophils incubated with strains whose cap-sules contained glucuronoxylomannan and galactoxyloman-nan have been demonstrated to produce neither ROS norNETs, but they did in non-capsulated strains. NET-associatedantimicrobial peptide, such as MPO, elastase and collage-nases, are needed to kill the fungus.61

ParasitesThe number of studies investigating the role of NETs inimmune responses toward protozoan parasites is increas-ing, although most of them have been conducted usinganimal-derived (e.g. goat or seal) neutrophils. Plasmodiumfalciparum induces NET formation. NETs entangle parasi-tized red blood cells and trophozoites together with anti-nuclear antibodies are involved in the pathophysiology ofmalaria in children and in the development of autoimmunephenotypes.62

The two main parasite stages of Leishmania, amastigotesand promastigotes, have been found to induce NETs ex vivo,which acts as a mechanism of defence against the infection.The induction of NETs is independent of NOS activity and ROSrelease. The entanglement of parasites within NETsdecreased the viability of parasites, even if some authorsstate that the main role for NETs is immobilization of para-sites and control of the infection.63,64

Toxoplasma gondii can stimulate NET formation. Thepresence of the parasite in the bloodstream is not necessaryto trigger NET release. NETs kill around 25% of the entangledparasites, thus controlling the infection. In human blood-derived neutrophils, the production of NETs has beendemonstrated by the activation of the Raf/MEK/ERK pathwayin response to T. gondii.65

Finally, some animal parasites, such as Eimeria bovis andBesnoitia besnoiti, were reported to participate to NETformation.66–69



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NETs in Cardiovascular Diseases

Dysregulation of neutrophil function has been recentlyindicated to play a pivotal role throughout the pathogenesisof most representative vascular diseases, such as acutecoronary syndrome (ACS), stroke and venous thrombosis.Among these disorders, ACS and stroke are dramatic com-plications of advanced atherosclerosis. In the earliest phasesof atherogenesis, neutrophils are recruited by upregulatedadhesion molecules on the dysfunctional endothelial sur-face, where they exacerbate the oxidative stress and invadethe vessel wall. After extravasation, and later in advancedatherosclerotic lesions, these cells sustain a vicious cycleleading to chronic inflammation and increased plaque vul-nerability by releasing oxidative enzymes, ROS and chemo-kines.70 On the contrary, during venous thrombosis,neutrophils are largely recruited from activated endotheliumthrough vonWillebrand factor (vWF)-, tissue factor (TF)- andadhesion molecule-mediated pathways accounting for thelarge part of inflammatory cells found in thrombus duringthe early stage of the disease.71 Thus, after the description ofNETosis in 2004, questions raised about its specific contribu-tion to the pathogenesis of CV disease and, consequently, itsfeasibility as marker of disease or therapeutic target.

NET Activity in CV Risk Factors and Early EndothelialDysfunctionSupporting the pathophysiological link betweenNETosis andvascular diseases, some known CV risk factors are associatedwith exacerbated or dysfunctional NET production. Diabeticpatients showed high plasma levels of NET-associated pro-teins (e.g. elastase, mono-oligonucleosomes and double-strand DNA)72 and impaired NET release in response tobacterial infections.73 The constitutive NET formationdescribed during hyperglycaemia can blunt their formationin response to proper stimuli.74 Interestingly, this phenom-enon does not appear as a consequence of an impairedglycaemic control, rather it seems related to the chronicproinflammatory environment accompanying diabetes.75

Recently, age-related CV dysfunction has also been directlylinked toNETosis inmice.Whilewild-type oldmice showed ahigh rate of heart fibrosis and a decline in systolic anddiastolic ventricular function, these features are consider-ably reduced in age-matched PAD4�/� animals, in whichNETosis is abolished.76 Similarly, hypertension has beendemonstrated to induce NETosis and platelet recruitmentin wild type, but not in PAD4�/� or DNase-treated myocar-dium.76 On the contrary, endothelial dysfunction is widelydescribed as primum movens of atherogenesis. NET-asso-ciated proteins have already shown their ability to induceendothelial toxicity and increase its thrombogenicity. Bothcirculating cell-free ds-DNA and histones are released byNETs upon the effect of inflammation and, acting as danger-associated molecular patterns (DAMPs), they can exacerbateinflammation itself, thus creating a deleterious vicious cycleat the endothelial level.77,78 In particular, histones have beenshown to stimulate the exocytosis of proinflammatory andprocoagulant Weibel-Palade bodies by endothelial cells.79

Moreover, matrix metalloproteinase (MMP)-9 containedwithin the NET web can reduce aortic endothelium-depen-dent vasorelaxation and trigger endothelial dysfunctionthrough the activation of MMP-2.80 Interestingly, interac-tions between NET and dysfunctional endothelium are notunidirectional: activated endothelial cells are able to induceNET formation in a C-X-C motif chemokine (CXCL)8-depen-dent way and extensive neutrophil co-culture with endothe-lial cells results in endothelial damage, which could beabrogated by DNase or through the inhibition of NOX.81

The Role of NETs in Atherosclerosis andAtherothrombosisNETs have been identified within atherosclerotic lesions andarterial thrombi in both human samples and atheroscleroticanimals.82,83 The pathophysiological relevance of NETosis inatherosclerosis has been underscored by inhibiting PAD4 inApoE�/�micewith chloramidine.84 Inparticular, chloramidine-treated mice showed reduced vessel inflammation (e.g. inter-feron [IFN]-α levels), plaque size and thrombotic attitude.84

Recently, interesting insights in this field came from a researchbyWarnatschet al.85 In this study, cholesterol crystalswereableto induce NETosis in vivo and NETs could primemacrophage toproduce pro-IL-1β, thus activating a strong pro-inflammatoryTh17 response.85 In atherosclerotic-prone mice fed with highcholesterol diet, intraplaque NETs co-localizedwith cholesterolcrystals andmacrophages near the necrotic core,while they didnot in more stable zones. The same atherosclerosis model,when lacking NE and proteinase-3, developed dramaticallysmaller and less inflamed atherosclerotic lesions with respectto ApoE�/� control mice, together with lower plasma IL-1βlevels.85 Another mechanism by which NETs are supposed toincrease atherogenesis has been described by Döring et al andinvolves plasmacytoidDC.86 In this paper, complexes formedbyextracellular DNA and neutrophil-derived proteins have beenshownto increase theplaqueburdenbystimulatingaDC-drivenstrong type I IFN response.On the contrary, the absenceof thesecells leads to decreased atherogenesis and reduced inflamma-tory response.86

Preclinical studies specifically investigating theeffectofNETmodulation on atherothrombosis are scarce and the currentknowledge largely derives from immunohistochemical analy-sis of human specimens. The blockage of neutrophil-derivednucleosomes by anti-histones antibodies treatment has beendemonstrated to prolong the time to occlusion in the ferricchloride arterial injury model.87 This effect was completelyreversed in elastase/cathepsin G double knockout mice; undera mechanistic point of view, nucleosomes are able to facilitatethrombosisbyallowingNEtodigest theTFpathway inhibitor.87

Moreover, neutrophil-derived serine proteases and nucleo-somes, two main components of NETs, may contribute toarterial thrombosis in the context of sterile inflammationsupporting dramatic atherosclerosis complications, such asmyocardial infarction and ischemic stroke. In a mouse modelof myocardial ischaemia/reperfusion (I/R) injury, NETosis tar-geting via DNase I injection has shown cardioprotective fea-tures by reducing both plasma levels of nucleosomes andcitrullinated histone 3 (citH3) presence at the site of injury.88



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Similar featureshavebeenfoundaftercardiac ischemicdamagein PAD4�/� mice.88 Finally, concurrent DNase I and recombi-nant-tissue plasminogen activator (r-TPA) therapy has beendemonstrated to reduce infarct size, no-reflow area and post-ischaemic left ventricle remodelling in an animalmyocardial I/R injurymodel, whereas these beneficial effects have not beenobserved in rats treated with DNase and r-TPA alone,89 thussuggesting a potential role for anti-NET agents as therapeuticstrategy during ischaemic heart diseases.

In human coronary specimens from infarcted patients,NETs were most frequently found in the early stages ofthrombus evolution (e.g. fresh and lytic thrombi), whilethese cells missed in advanced, organized thrombi.83

Recently, a study analysing coronary thrombectomy frompatients with ST-elevatedmyocardial infarction showed thatthe thrombus NET burden correlates in a positive fashionwith the infarct size and negatively with ST-segment resolu-tion.90 In the same work, the plasma collected from theculprit lesion site contained higher concentrations of NET-related biomarkers when compared with femoral samplesfrom the samepatients aswell as DNase activity at the culpritlesion site negatively correlated with thrombus NET burdenand infarct size. Moreover, recombinant DNase acceleratedthe coronary thrombi lysis in vitro.90 In particular, Stakoset al recently demonstrated that neutrophils at culprit lesionsite can release thrombogenic signals through NET formationand subsequent delivery of active TF.91

Even if most of the currently available evidence linksNETs to atherothrombosis, they may also participate toearlier superficial erosion of the plaque by inducingendothelial cell apoptosis.92 Of importance, biomarkers ofNETosis already showed to be positively associated with theseverity of atherosclerosis and to predict future CV events(►Table 2). Under this point of view, Borissoff et al gavefundamental insights by correlating the disease severity inpatients with coronary disease assessed by computedtomographic angiography and markers of NETosis.93

Although these data underline the importance of suchdeath mechanism in atherosclerosis-related diseases,more studies are needed to overcome the low specificityof some NET-related biomarkers (e.g. histones and doublestrain [ds]DNA).

NETs have also been investigated in the setting of acuteischemic stroke (AIS) highlighting the role of thrombo-inflammation as a pivotal player in the pathophysiology ofischaemic stroke94 along with thrombosis and inflamma-tion, which constitute a loop of bidirectional regulationcontributing to ischaemic damage in brain or other tissues.95

Recently, Vallés et al described for the first time that threemarkers of NETs—dsDNA, nucleosomes and citH3—are sig-nificantly elevated in patients with AISwhen compared withhealthy subjects; in particular, the greatest proportionalincrease was found for the most specific NET markercitH3.96 Levels of the above-mentioned NET markers havebeen correlated with stroke severity at onset and dischargeby the clinical National Institutes of Health Stroke Scale andmodified Rankins scale scores as well as by significantelevations of citH3, dsDNA and nucleosomes. Authors also

found that citH3 and dsDNA levels were higher in patientswith cardioembolic stroke, this being related to a higherinflammatory activation. This issue was in touch with thesignificant increase in NET markers for patients with ahistory of atrial fibrillation (AF), too. Finally, citH3 hasbeen found elevated especially in older patients, withhigher fasting glucose, and with prior AF and independentlyassociated with all-cause mortality at 1-year follow-up.96

These results indicate that citH3 might represent a usefulprognostic marker in patients with AIS, warranting newresearches for neuroprotective therapies in this field.The results of other studies investigating NETs in AIS arelisted in ►Table 3.

NETs and Venous ThrombosisAlthough arterial and venous thromboses are different syn-dromes with different main causes, they found in NETs acommon pathogenic pathway. Several key components ofNETs, such asnucleic acids, histones and enzymes, have shownvenous procoagulant features.97–99 Histone infusion leads tovWF release and has been found to accelerate the thrombusformation in inferior vena cava (IVC) stenosis model of deepvenous thrombosis (DVT).100 Thrombi from the same animalmodels are characterized by an important presence of NETsassociated with vWF, especially in the earliest stages.100,101

NETs interact with vWF through histone A1 domain,102 butthey can also bind other thrombosis-related proteins (e.g.fibronectin) containing several DNA-binding domains.101

Not only vWF but alsoTF sustains the strong relation betweenvenous thrombosis and NETs. During the generation of NET,neutrophils produce both TF and NE. NE is critical to furtherincrease TF activity by the cleavage of TF inhibitory mole-cules.71,91 Thus, after platelet and neutrophil activation byprocoagulant factors, the latter generates NETs acting as trapand framework for thrombuselements, such as red blood cells,leukocytes, platelets and activated coagulation factors.71,103

The crucial contribution of NETosis in venous thrombosis hasbeen underlined by PAD4�/� mice, in which the lack of NETsresults in fewer thrombi after IVC stenosis compared withwild-type ones; similar effects have also been demonstratedby the administration of DNase I.104 Although some contro-versies still remain,105 these results encouraged scientists toshift their attention from preclinical setting to the bedside. In2013, two papers showed an association between increasedplasma markers of NETs and DVT (►Table 2). The first articlestated that in patients with DVT circulating nucleosomes andelastase-α1-antitrypsin complexes, levels are increased withrespect to patients with first clinical suspicion of DVT, whichhas been then excluded by ultrasonography.106 The secondstudy focused on plasma DNA level showing higher concen-trations of thismarker in DVT patients and its correlationwithD-dimer,Wells score andMPO, the third indicating neutrophilas the source of the nucleic acid.107 Recently, the immunohis-tochemical analysis of thrombi at different stages of develop-ment fromhumansurgeryor autopsy showedhighpresenceofNETs (indicated by the association of citrullinated histone H3with MPO and DNA) in thrombi during the phase oforganization.108



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Table 2 Recent studies investigating NET biomarkers in CV diseases

Author Year Patients Biomarkers Results

Atherothrombosis

Shimony et al231 2010 16 patients with STEMI and47 healthy subjects

dsDNA dsDNA levels were significantlyhigher in patients compared withcontrols (p ¼ 0.001) and positivelycorrelated with levels of CK and TnT(r ¼ 0.79 and 0.65, p < 0.001 andp ¼ 0.006, respectively)

Borissoff et al93 2013 282 patients with suspectedcoronary heart diseaseundergoing coronary CTAwere grouped according tothe presence and severity ofCAD

dsDNA,nucleosomes,citH4 and MPO-DNAcomplexes

dsDNA, nucleosomes and MPO-DNAcomplex levels were significantlyhigher in patients with severe CAD(p ¼ 0.003, p < 0.001 and p < 0.05,respectively) or abundant coronaryartery calcification (p < 0.001 for all)with respect to healthy controls.Their levels correlate with theseverity of luminal stenosis(p � 0.001 for all) and with numberof diseased coronary artery segments(p � 0.001 for all).Baseline values higher than the totalgroup median of dsDNA (OR, 3.12;95% CI, 1.27–7.63; p ¼ 0.013),nucleosomes (OR, 2.59; 95% CI,1.09–6.14; p ¼ 0.030) andMPO–DNA (OR, 3.53; 95% CI,1.38–9.03; p ¼ 0.009) were signifi-cantly associated with the occurrenceof MACEs

Cui et al232 2013 137 ACS patients (51 withunstable AP, 37 with NSTEMI,and 49 with STEMI), 13 stableAP patients, and 60 healthycontrols

dsDNA ACS patients showed higher dsDNAlevels compared with stable APpatients and control group (p < 0.05for both). Significant differences indsDNA concentrations wereobserved in ACS group amongunstable AP, NSTEMI, and STEMIsub-groups (p < 0.05 for all).dsDNA levels were different amongACS patients divided into threegroups according to Gensini scorewith increasing levels of dsDNAconcurrent with increasing Gensiniscore (p < 0.05, for all)

Mangold et al90 2015 111 patients with STEMIundergoing PCI

Nucleosomes anddsDNA

Nucleosomes and dsDNA levels weresignificantly higher at the culpritlesion site than to the femoral artery(p ¼ 0.0002 and p < 0.0001,respectively)

Helseth et al233 2016 30 patients with CAD under-going PCI (20 with STEMI and10 with stable AP)

dsDNA andnucleosomes

dsDNA and nucleosome levels werehigher in patients with STEMIcompared with patients with AP(p < 0.03 for both). dsDNA signifi-cantly correlated with peak TnT andCK-MB at day 5 (p ¼ 0.03) and withlesion size assessed by MRI at days 5and 7 (p ¼ 0.01 and 0.04,respectively). Nucleosomes correlatewith infarct size at day 5 (p ¼ 0.02)

(Continued)



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NETs in Autoimmune and AutoinflammatoryDiseases

Systemic autoimmune diseases develop as multistep pro-cesses through a complex interplay between genetic andenvironmental factors leading to cellular damage and theconsequent exposure of immune cells to autoantigens. Theimbalance of different cell death mechanisms (apoptosis,necroptosis, pyroptosis, NETosis and autophagy) has beenproposed as a critical source of modified and/or externalizedautoantigens.109 Especially nuclear material released fromNETs seems to bemore immunogenic than the apoptotic one.Both native and oxidized self-DNA bound to NETs activateDCs to synthetize IFN-α in a TLR-dependent manner.110,111

In mice, the immunization with NET-loaded DCs promotesthe development of autoimmunity better than apoptoticneutrophil debris.112 NETs also increase T-cell response toantigens and activate B cells to induce immunoglobulin (Ig)class switching and antibody production.113 Once externa-lized, oxidized DNA is alsomore resistant to degradation andthis behaviour contributes to sustain a dysregulated immuneresponse.114 In addition, NET-mediated activation of theinflammasome further amplifies the inflammatory responsethrough a feed-forward loop. The inflammasome stimulationtriggers synthesis and release of IL-18 and IL-1β, which inturn induces NET formation.115 Activation of classic andalternative pathways of complement system as well ascoagulation cascade is additional immunogenic mechanisms

linking NETs to autoimmune/autoinflammatory diseases.116

Noteworthy, many of the proteins found in NETs are recog-nized as major autoantigenic targets in rheumatologic dis-eases: dsDNA and histones in SLE, vimentin and enolase inRA, MPO and proteinase-3 in vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Different auto-antibody profiles may then be influenced by the cargoprotein within NETs and detailed analysis revealed differ-ences in protein content and posttranslational modificationsassociated with different autoimmune diseases.117–119

Systemic Lupus ErythematosusNeutrophils from patients with SLE showed various abnorm-alities in their phenotype and function.120 Circulating levels ofapoptotic neutrophils, which may provide excess autoantigensuch as dsDNA, are increased inpatientswith SLE and correlatewith disease activity.121 Furthermore, patients with SLE arecharacterizedbyadistinct neutrophil subpopulation knownaslow-density granulocytes (LDGs).122 Those cells are prone torelease proinflammatory cytokines and show enhanced NETformation. NETs released by LDGs contain high levels of auto-antigens and immunostimulatory molecules, such as LL37,MMP-9, and α- and β-defensins.123 LDGs are also enriched ofdsDNAandoxidizednucleic acids,which are strong inducers ofIFN-α and NOD-like receptor family pyrin domain-containing(NLRP)3 inflammasome.115,124 Finally, patients with SLE exhi-bit impairedNET clearance correlatingwithdiseaseactivity.125

As a result of overproduction and defective clearance of NETs,

Table 2 (Continued)


Venous thrombosis

van Montfoortet al106

2013 150 patients with sympto-matic DVT and 195 patientswith clinical suspicion of DVTin whom the pathology wasexcluded by US examination

Nucleosomes Nucleosome levels were significantlyhigher in patients with DVT(p < 0.001) and positively correlatedwith neutrophil activation in bothcase and control samples (p < 0.001for both). An increase in nucleosomelevels >80th percentile carried anincreased risk of DVT than levels�80th percentile after adjustmentfor potential confounders(OR: 3.0, 95% CI: 1.7–5.0)

Diaz et al107 2013 47 patients with sympto-matic DVT, 28 patients withclinical suspicion of DVT notconfirmed by US examina-tion, and 19 healthyvolunteers

dsDNA dsDNA levels were higher in DVTgroup with respect to both negativecontrol groups (p < 0.01 for both).dsDNA levels showed a positivecorrelation with CRP (p < 0.01),D-dimer (p < 0.01), and vWF(p < 0.01) and the Wells score(p < 0.01). A negative correlationwas found with ADAMST13(p < 0.01)

Abbreviations: ADAMST13, a disintegrin and metalloproteinase with thromboSpondin-1 motifs (13th member of the family); CAD, coronary arterydisease; citH4, citrullinated histone H4; CK-MB, creatine kinase, muscle and brain; CRP, C-reactive protein; CTA, computed tomography angiography;CV, cardiovascular; ds, double strain; DVT, deep venous thrombosis; HR, hazard ratio; LV, left ventricle; MACEs, major cardiovascular events; MI,myocardial infarction; MPO,myeloperoxidase; MRI, magnetic resonance imaging; NSTEMI, non–ST-elevatedmyocardial infarction; N/L, neutrophil tolymphocyte; OR, odds ratio; PCI, percutaneous coronary intervention; RR, relative risk; SINTAX, Synergy between Percutaneous CoronaryIntervention with Taxus and Cardiac Surgery; STEMI, ST-elevated myocardial infarction; TnT, troponin T; US, ultrasound; vWF, von Willebrand factor.



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Table 3 Studies investigating NET biomarkers in acute ischemic stroke


Rainer et al234 2003 88 patients with stroke-likesymptoms presenting to theED

nDNA nDNA concentrations within 3 h of symp-tom onset were higher in died patientsthan in those who survived at discharge(p ¼ 0.03) as well as in died patients withNIHSS scores >8 who survived to 6 mo(p ¼ 0.002). nDNA concentrations corre-lated with the volume of cerebral hema-toma (p ¼ 0.03). nDNA concentrationspredict 6-mo mortality (OR: 1.6, 95% CI:1.1–2.4; p ¼ 0.03) and 6-mo RS score >2(OR: 1.8, 95% CI: 1.0–3.3; p ¼ 0.05)

Geiger et al235 2006 63 patients with strokeobserved daily during thefirst week

Nucleosomes In patients with BI score �50, the increasein nucleosomes is prolonged until day 5.Patients with BI score <50 showed asteeper initial increase with a maximum onday 3. Both days after stroke and BI scoresignificantly influenced nucleosome con-centrations (p < 0.001 for both). Nucleo-some concentration showed a significantcorrelation on day 3 with infarction volume(p ¼ 0.001)

Lam et al236 2006 44 patients aged �18 ypresenting to the ED with astroke-like syndrome butnegative neuroimagingresults

nDNA Patients with post-stroke mRS grades 3–6have been shown with a nDNA concentra-tion significantly higher than that ofpatients with post-stroke mRS grades 0–2(p ¼ 0.01). nDNA concentration couldpredict post-stroke morbidity and mortal-ity in patients with negative neuroimaging

Geiger et al237 2007 63 patients with strokeobserved daily during thefirst week

Nucleosomes Levels of nucleosomes at days 3 and 6correlated significantly with initial BI(p ¼ 0.0023 and 0.0284, respectively) andwith infarction volume only at day 3(p ¼ 0.0001). Strong correlations havebeen shown between BI at admission andBI at discharge and between BI at admis-sion and infarction volume (p < 0.0001 forboth). In patients with initially severedefects (BI <50), nucleosomes at day 3have been found to be prognosticallyrelevant (p ¼ 0.014). In multivariateanalysis, nucleosomes and BI at admissionshowed independent prognostic relevance(p ¼ 0.039)

Tsai et al238 2011 50 AIS patients and50 control subjects

nDNA andmDNA

Levels of nDNA and mDNA were higher inpatients with AIS than in controls(p < 0.05). Elevated circulating nDNA inplasma persisted until 1 mo after AIS.Levels of nDNA positively correlated withthe clinical severity of stroke according toNIHSS score

Hirose et al239 2014 49 critically ill patientsadmitted to ICU, of whom8 with stroke

DNA, citH3 DNA has been found elevated in patientswith stroke with respect to controls. citH3has been detected in blood smears byimmunofluorescence

Thålin et al240 2016 31 patients withischemic stroke

DNA, citH3 Patients with concurrent hsTnT elevationrevealed cerebral micro-thrombosis withcitH3 in thrombi in a higher rate thancontrols (p < 0.001). citH3 correlatedpositively with thrombin–antithrombincomplex (p ¼ 0.004) and solubleP-selectin (p < 0.001)

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the presentation of autoantigens to autoreactive B cells isenhanced.116,126 A leading role in SLE pathogenesis may beplayedbyanalternative formofNETs, calledmitochondrialDNANETs. In fact, more severe forms of SLE have been observed inmice after injection of oxidized mitochondrial DNA and inhumanscarryingNOX-deficientgenes (typically inCGD).124,127

Although the roleofNETs inSLE requires further investigations,their high levels found in the skin, kidney and bone marrowsupport a direct role in SLE-associated organ dysfunction123

andpreliminaryclinical studies support a potential associationbetween circulating levels of NETs and disease activity(►Table 4).116,123,125,128–131

Rheumatoid ArthritisIn RA, activated neutrophils are themost abundant cells in thesynovial fluid. In addition, awide number of anti-citrullinatedproteins/peptides antibodies (ACPA) is produced in RA, repre-senting specific disease markers.132 Both endogenous andexogenous antigens become target of ACPA after deimination(or citrullination), which is a post-translational modificationlargely catalysed by the PAD2 and 4, usually overexpressed inRA patients.133,134 A major contribution to the citrullinationcomes from NET generated from activated neutrophils, espe-cially those belonging to the LDG subpopulation.118,135

Furthermore, deiminated histones have been recognized askey mechanism leading to ACPA generation in RA patients,especially those with Felty syndrome.59,136 Noteworthy, theectopic lymphoidstructures localized in theRA joint synoviummay also contribute to the NET generation. They representfunctional structures supporting the clonal selection of auto-reactive B cells and then their differentiation to plasma cellsproducing antibodies against citrullinated antigens.137–139

Therefore, a delay in the clearance of NETs might form areservoir of citrullinated antigens, which sustain the autoim-mune response in RA as already described for SLE.125

ANCA-Associated VasculitisANCA-associated vasculitis (AAV) is referred to as a group ofpauci-immune vasculitis characterized by neutrophil-richnecrotizing inflammation of small vessels and the presenceof ANCAs. In this context, NETs have been recently found at thesites of vasculitic lesions (kidneyand skin) and in thrombi, bothas co-localizations of DNA and granule proteins and as merecitrullinated histones.112,140–145 Some cross-sectional studiescompared NET levels during remission and active disease,although results remain inconclusive.146–148 Neutrophilsfrom patients with AAV are less prone to undergo apoptosisand show spontaneous NET formation. Even though the high

Table 3 (Continued)


Vallés et al96 2017 243 patients with AISfollowed up for 12 mo afterthe event

dsDNA,nucleosomes,citH3

dsDNA, nucleosomes and citH3 weresignificantly higher among patients withAIS compared with healthy subjects(p < 0.05; p < 0.001 for the latter). Theseparameters were increased in patients whowere older than 65 y (p < 0.001), in thosewith a history of AF (p ¼ 0.013 for dsDNA,p ¼ 0.007 for citH3, p ¼ 0.02 for nucleo-somes), CE stroke (p < 0.05 for dsDNA andcitH3), high glucose levels (p < 0.05 for allmarkers) and severe stroke scores atadmission (p < 0.001 for all markers) anddischarge (p < 0.001 for citH3 andnucleosomes, p ¼ 0.038 for dsDNA). Inmultivariate analysis, elevated levels ofcitH3 at onset was independentlyassociated with AF (OR: 6.704, 95% CI:1.4–32.1; p ¼ 0.017) and with all-causemortality at 1-year follow-up (OR: 7.055,95% CI: 1.631–30.50; p ¼ 0.009)

Laridan et al241 2017 68 ischemic stroke patientsundergoing endovasculartreatment

DNA, citH3 citH3 was observed in almost all thrombiand co-localized with extracellular DNAreleased from neutrophils. citH3 was moreabundant in thrombi of CE origin com-pared with other etiologies (p < 0.05).Older thrombi contained significantlymore neutrophils and citH3 comparedwith fresh thrombi (p < 0.001 andp < 0.05, respectively)

Abbreviations: AF, atrial fibrillation; AIS, acute ischemic stroke; AUC, area under the curve; BI, Barthel index; CE, cardioembolic; CI, confidenceinterval; citH3, citrullinated histone H3; CRP, C-reactive protein; ds, double strain; ED, emergency department; hsTnT, high sensitive troponinT; mRS,modified Rankin scale; NIHSS, National Institutes of Health Stroke Scale; m, mitochondrial; n, nuclear; NET, neutrophil extracellular trap; NSE,neuron-specific enolase; OR, odds ratio.



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fraction of LDGs observed in AAV may explain this behaviour,normal-density neutrophils have also been found to sponta-neously release more NETs as compared with healthy blooddonors.149 A growing body of data indicates ANCAs not only asneutrophil activators but also as promoters of NET generation.As neutrophil activation is epitope specific, epitope specificityandaffinityare found increasedduring activedisease.150–152 Inturn, the overproduction of NETs enhanced the exposition ofepitopes (MPO end proteinase-3), further perpetuating thegeneration of ANCAs.129,140 Alongside, elevated levels of NETsin AAV patients may be explained by a reduced NET clearanceas confirmed by in vitro experiments.129

Other Autoimmune DiseasesRecent evidence links NETs and antiphospholipid (aPL) anti-body syndrome (APS). High levels of dsDNA and NETs have

been found in patients with APS and the serum of thosepatients display defective NET degradation.153,154 More spe-cifically, aPL antibodies frompatientswithAPSmay induce therelease of NETs from control neutrophils and especiallyLDGs.154 Also, thrombi from mice treated with APS IgG havebeen recently found to be enriched of citrullinated histoneH3,thus indicating a role for NETs in thrombotic complications ofAPS.155 Further supporting a direct role in thrombosis, grow-ing data linked NETs to preeclampsia, also in aPL-negativepatients.156–158 Finally, NETosis has also been observed inpsoriatic skin lesions. By inducing the expression of humanβ-defensin-2 in keratinocytes, NETs amplify the local inflam-mation leading to DC activation and consequent developmentofMunro’s abscess.159,160 Finally, a direct correlation betweenthe amount of NETs in the peripheral blood and diseaseseverity has also been demonstrated.160

Table 4 Clinical evidence linking circulating NETs to clinical activity of autoimmune diseases

Author Year Disease Patients Outcome Result

Hakkim et al125 2010 SLE Lupus nephritis vs.healthy controls

Activity index onrenal biopsy

Poor NET degradation was associatedwith lupus nephritis and increasedserum levels of anti-dsDNA antibodies

Villanuevaet al123

2011 SLE III or IV class GN Activity index onrenal biopsy

Patients with class IV GN had higheractivity index (% of netting neutrophils)and higher circulating levels ofanti-dsDNA antibodies

Leffler et al116 2012 SLE Patients with diseaseremission andflare vs. healthy controls

Disease activity Low NET degradation was associatedwith complement activation/deposi-tion, high levels of circulating autoanti-bodies and renal involvement (GN)

Leffler et al128 2013 SLE 69 patients followed upfor a median of 784 d

Disease activity Decreased ability to degrade NETs wasassociated with clinical manifestationsin SLE according with the SLEDAI-2Kscore

Nakazawaet al129

2014 MPASLE

38 patients withMPA vs. 23 SLE vs. 8control subjects

BVASSLEDAI-2K score

Both MPA and SLE patients showedreduced NET degradation. In MPApatients, this ability correlated withdisease activity, while no correlationwas shown between NETs and diseaseactivity (SLEDAI-2K score) in SLEpatients

Zhang et al130 2014 SLE 54 patients vs. 43control subjects

Lupus nephritis High circulating levels of cfDNA (markerof NET dysregulation) was found inpatients with SLE and were associatedwith markers of renal injury, such as24-h urinary protein content (r ¼ 0.350;p ¼ 0.013), serum albumin (r ¼� 0.500; p < 0.001), and creatinineclearance (r ¼ � 0.354; p ¼ 0.044)

Pérez-Sánchezet al131

2017 RA 106 patients vs. 40control subjects

RF, ESR, CRP,NO, cIMT

RA patients exhibited enhanced NETgeneration and impaired DNase activity.Furthermore, NETosis-derived products,such as cfDNA, correlated with autoim-mune parameters, inflammatorymediators, oxidative stress markers aswell as early atherosclerosis

Abbreviations: BVAS, Birmingham vasculitis activity score; cfDNA, cell-free DNA; cIMT, carotid intima media thickness; CRP, C-reactive protein;dsDNA, double-stranded DNA; ESR, erythrocyte sedimentation rate; GN, glomerulonephritis; NET, neutrophil extracellular trap; NO, nitric oxide;MPA, microscopic polyangiitis; RA, rheumatoid arthritis; RF, rheumatoid factor; SLE, systemic lupus erythematosus; SLEDAI-2K, Systemic LupusErythematosus Disease Activity Index 2000.



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Gout and Inflammatory Bowel DiseasesActually, very little is known about the link between NETs andgout and inflammatory bowel diseases (IBDs), such as Crohn’sdisease (CD)andulcerative colitis (UC). Ingout,NETshavebeendescribed not only to feed inflammation,161 but also to reg-ulate the inflammatory process and block gout episodes.162

NET generation is responsible for the reduction of neutrophildensity, then DNA nets encapsulate monosodium urate crys-tals protecting them from further phagocytosis, and finallyNET-derived proteases block cytokines.163,164

NET formation has been poorly studied in IBDs. As ROSlevels are elevated, it is likely that neutrophils could produceNETs. In UC, NETs have been observed and correlated withinflammation by proteomic studies, but more studies arewarranted to clarify the NET involvement in both CD andUC.165 In 2016, He et al evaluated the role of NETs in IBDs. Theauthors found that NETs were generated by peripheral bloodneutrophils from patients with active IBD; however, stimula-tion with sera from patients with active disease also inducedsignificantly NET release on neutrophils isolated from healthysubjects. Moreover, NETs along with phosphatidylserine (PS)exposure on platelets, leukocytes and endothelial cells areinvolved in the hypercoagulability characterizing active IBD.The researchers also proved an inhibitory effect of lactadherinandDNase I onPS andNETs, separately, suggestingnew targetsfor IBD drugs in next studies.166

NETs and Lung Diseases

In the last decade, the formation of NETs has drawn greatattention concerning lung diseases. Components of NETshave been shown to damage both epithelial and endothelialcells as well as connective tissue worsening the lung pathol-ogy.167 Indeed, NETs have been identified in the lung ofpatients suffering from cystic fibrosis (CF), transfusion-related acute lung injury (TRALI), asthma, chronic obstruc-tive pulmonary disease (COPD), and in lungs infected withbacteria, virus, or fungi.168 In these pulmonary diseases,NETs have been described as emerging pathophysiologicalplayers of potential therapeutic interest.

Cystic FibrosisIn CF, patients with high levels of free DNA in the sputumhave been shown with a diminished lung function as com-pared with those with mild disease, because the airwayobstruction is mainly a consequence of the amount ofNETs and DNA.161 Marcos et al recently confirmed thisfinding, showing that free CF airway DNA levels correlatedwith pulmonary obstruction both in CF patients andmice.162

The importance of NETs in CF is highlighted by the fact thatthe elimination of free DNA from patient’s airways is con-sidered as an important therapeutic option. In fact, in bothearly and mild stages of the disease, NETs can provideextracellular antibacterial and antifungal host defences. Atthis time, recombinant inhaled DNase should be used morecautiously, being potentially responsible for the delivery ofencaptured pathogens. Differently, in moderate to severestages of CF, the amount of mucus and DNAwas responsible

for airway obstruction, which can be efficiently resolved byDNase by cleaving DNA traps.162,169 Moreover, isolates ofPseudomonas aeruginosa can trigger a great respiratory burstand NET release in CF.170 P. aeruginosa-mediated NET for-mation was found responsible for the bactericidal perme-ability-increasing (BPI) protein cleavage by P. aeruginosaelastase, suggesting a novel mechanism in the developmentof autoimmunity to BPI.171 Moreover, the authors alsoprovided a role for autoimmunity in CF disease severity, asautoantibody levels have been associated with diminishedlung function.

Chronic Obstructive Pulmonary DiseaseNeutrophil elastase has been detected in the airway mucosaof COPD patients during severe exacerbations showing aproinflammatory role via the secretion of CXCL8, which isa powerful NET inducer.172 In the sputum from acutelyexacerbated COPD patients, NETs and NETotic neutrophilshave been found in a great amount by confocal laser micro-scopy and electron microscopy; an abundance of NE andcitH3 has also been demonstrated.173 These findings witnessthat NETosis can be considered as a part of COPD pathology,relevant for new therapeutic options. Anyway, NETs havebeen described in the airways of stable COPD patients as amarker of neutrophils in the sputum.174,175 In particular, apositive correlation between the abundance of NETs in thesputum of COPD patients and disease severity has beendescribed; in fact, more than 90% of exacerbated COPDpatients presented with a higher number of NETs in theirsputum with respect to stable COPD patients. Indeed, theNET amount directly correlated with the disease severity interms of airflow limitation.176 Recently, levels of NETs in thesputum of COPD patients have been directly associated withthe severity of the disease and the number of exacerbationsas well as with the loss of microbiota diversity and impairedex vivo neutrophil phagocytosis.177 In this view, NETs couldbe an interesting therapeutic target in the future, consider-ing that the only effective drugs are currently long-actingbronchodilators, which do not affect inflammation.

AsthmaAsthma has been classically considered as an eosinophilicdisease. However, recent data claim that some asthmaticpatients show an important neutrophilic inflammation inthe lungs. Patients with “neutrophilic” asthma have shown areduced response to the classical therapy with glucocorti-coids,which in turn aggravate local inflammation by increas-ing neutrophil survival.178

In addition, glucocorticoid administration to neutrophilicasthmatics could aggravate lung inflammation, since gluco-corticoids can prolong neutrophil survival. Since the hypoth-esis of autoimmune involvement in asthma has gained greatinterest recently, NETs have been demonstrated as keyplayers in the stimulation of airway epithelial cells to pro-duce autoantigens, especially in severe asthma, as antibodiesagainst NE or MPO attenuated these effects.179 Dworski et alhave shown that eosinophils in the airways of atopic asth-matic individuals could release eosinophil ETs and co-



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localized with eosinophil granule proteins, such as majorbasic protein and eosinophil cationic protein.180 In this case,DNA was of mitochondrial origin, and not nuclear.181 Inter-estingly, allergens did not showany increase in eosinophil ETor NET formation in the airways of asthmatic patients.180

Recently, eosinophils from asthmatic mice have beendemonstrated to release eosinophil ETs co-localizing witheosinophil peroxidase aggravating pulmonary impairment,which was reversed by DNase therapy.182 Recombinanthuman DNase treatment improved resistance and decreasedoxidative stress in the lungs of asthmatic mice.183 In light ofthis, a combined use of recombinant human DNase therapyalong with inhaled glucocorticoids may provide a reductionin sputum viscosity and improve the quality of life andprognosis of these patients.

Transfusion-Related Acute Lung InjuryTRALI is the leading cause of blood transfusion-related deathdeveloping within 6 hour of transfusion and presenting withhypoxemia, respiratory distress, and pulmonary infiltrates.In 2012, Thomas et al demonstrated that NETs form duringTRALI both in humans and inmice and that their degradationby DNase 1 inhalation improved the condition of mice withTRALI.184 Also, platelets can accumulate in the lungs of micewith TRALI and have been described to induce NET forma-tion.185 In turn, histones expressed by NETs may activateplatelets, thus feeding a vicious cycle.186 As a further proof ofit, the pre-treatment of mice with a histone-blocking anti-body decreased NET generation as well as lung oedema, lungvascular permeability, and mortality.185

NETs and Cancer

Neutrophils are known to be present inside and around solidcancers since long time, thus being the most representativetumour-infiltrating immune cells. Anyway, controversieshave been raised about their role in this setting and twosubsets of tumour-associated neutrophils (TANs) have beenfound to develop depending on the influences of the tumourmicroenvironment: the N1 phenotype displays proinflam-matory and antitumourigenic functions, while the N2 phe-notype has protumorigenic activity with transforminggrowth factor-β produced by the local tumour microenvir-onment playing a central role in polarizing mature neutro-phils to adopt a pro-tumour N2 phenotype.187 Manyquestions are open about NET’s function in tumour growthmodulation, even if a role for NETs has been recognized in thelocal tumour development and especially in tumour-asso-ciated thrombosis.

The first finding of a role for NETs in tumours dates backto 2013 based on a small number of Ewing’s sarcomasamples suggesting that patients with intratumoral NETsexperienced a poorer prognosis.188 A stronger suggestionfor a role of NETs in tumour progression comes fromstudies investigating NET-associated proteins, such as NEand MPO. NE can directly impact on tumour growth,progression, and cell migration by inducing cell prolifera-tion in both human and mouse adenocarcinoma cell

lines.189 In addition, NE deficiency has been found to blunttumour burden in mice.189 Although these results mainlyreferred to the soluble NE, they suggest a potential role forNETs, which need to be clarified by targeted study. Sanga-letti et al found that NETs could stimulate the proliferationand malignant transformation of B cells toward malignantlymphoma via the NF-κB signaling.190 A role for NETs hasalso been tested in pancreatic ductal adenocarcinoma.191

Another NET-associated protein involved in the pathophy-siology of cancers is MMP-9, which contributes to carcino-genesis, tumour growth and progression, and metastasis.Apart from its role in different cancer types, it is stillcontroversial whether NET-bound MMP-9 conveys theseeffects or NETs can even protect fromMMP-9 functions; thereason of this controversy can be found in the fact thatMMP-9 has been shown to be inactive when bound toNETs.192 Since IL-8 has been demonstrated to play a role inNET generation and angiogenesis, a clear clinical relevancefor IL-8 in tumour progression has been shown for manytumours, too.193,194 The cascade including IL-8 can beprobably considered a vicious cycle started by NETs them-selves recruiting additional neutrophils, which in turnproduce NETs, all this ultimately concluding with tumourgrowth and angiogenesis. Demers et al have recently linkedtumour progression and NETosis by showing that primingof neutrophils by tumour-associated granulocyte-colonystimulating growth factor (G-CSF) is able to promotetumour growth. Indeed, PAD4-deficiency was protectiveonly against tumour progression when the implantedtumour cell line produced G-CSF.195

Various studies have confirmed a role for TANs in theenhancement of cancer cell survival, migration and poorprognosis, but none of this has focused on tumour-associatedNETs.196–198 Interestingly, in vivo evidence from cutaneousmelanoma has shown that it becomes more aggressive andmetastatic after ultraviolet radiation because of neutrophilrecruitment. In fact, neutrophil recruitment has been asso-ciated with a more migratory phenotype, local angiogenesisand angiotropism of melanoma cells,199 with a possibleresponsibility for NETosis, although NETs have not beenspecifically focused.

CXCL8 can also play a role in tumour-associated throm-bosis as it is contained in microparticles. In the granulocyticsubset of myeloid-derived suppressor cells (considered animportant T-cell immunosuppressive component in cancer-bearing hosts), IL-8 has been shown to stimulate the forma-tion of NETs.200 Interestingly, tumour-derivedmicroparticlescan interact with macrophages, activating them, and pro-mote the production of tumour necrosis factor-α favouringthe recruitment of other inflammatory cells and NETs.201

vWF is a known actor in metastasis and tumour growthand the interaction with NETs can result deleterious. In micemodels, vWF null mice or mice treatedwith antiglycoproteinIbα experienced increased experimental lung metasta-sis.202,203 NETs have been demonstrated to bind to vWF onthe vessel wall and this is important when considering thatmelanoma cells can stimulate endothelial cells to producevWF contributing to thrombosis.204



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In 2016, Guglietta et al investigated about the role ofcomplement in NET induction and tumour growth. In aspontaneous small intestine cancer model, hypercoagulationcan directly affect neutrophil effector function and is linked tocomplement activation, in particular C3a, showing anincreased number of TANs and low-density neutrophils.LDGs displayed features of N2 neutrophils and spontaneouslyunderwentNETosis,whichwasdependenton the involvementof the complement receptor C3aR. To block this reaction, theimmune system triggers the development of neutrophils withanN2 phenotype, which are responsible for tumour growth ina mutation-dependent protumorigenic milieu.205

A role for NETs has been hypothesized for metastasis, too,even if specific studies are still lacking. Neutrophils per sepromote the blocking of circulating tumour cells, especiallyunder inflammatory conditions, and NETs are very importantin this setting.206 Anyway, even considering other studies withDNase I suppressing tumorigenesis, the activation of periph-erals cells as a result of tumour-induced intravascular NETformation, which predisposes formetastasis, is still amatter ofdebate. Platelet–granulocyte complexes with tumour cells not

clearly identified as NETs have been proved to create earlymetastatic niches, which are pivotal for later metastatic pro-gression.207 Upon the secretion of CXCL5 and CXCL7 by plate-lets due to contact with platelets and the release of IL-8 fromtumour cells recruiting neutrophils, the tethering of tumourcells to endothelium takes place, leading to transendothelialmigrationfirstandthefollowingdevelopmentofmetastasis.208

Main roles of NETs in cancer are summarized in ►Fig. 2.

Future Perspectives

Accumulating data on the role of NETs in highly prevalentinflammatory conditions (such as sepsis, CV diseases, auto-immune and inflammatory diseases) pave the way to novelbiomarkers and treatments, which might readily becomeavailable for patient care. However, their translation into theclinical practice requires further investigations specificallyfocused on NET biology and measurement assay. First, mole-cules integrated in NETsmay vary according to environmentalfactors, so that the characterization of NET proteome repre-sents an exciting challenge for the next future. Second, the low

Fig. 2 The complex interplay between neutrophils and cancer. (A) Interactions between neutrophils and tumour cells lead to neutrophilextracellular trap (NET) production, which influence tumour growth and progression, angiogenesis and metastasis. In fact, tumour cells canrecruit and activate neutrophils, acquiring pro- and antitumoural properties. Neutrophils are also responsible for cytokine secretion promotingthe formation of NETs, which present a variety of antimicrobial and cytotoxic substances relevant for tumour growth and progression. (B) NETsmay influence the local growth and progression of cancer. Neutrophil elastase (NE) is known to stimulate tumour growth, progression andspreading. Interleukin (IL)-8 is responsible for angiogenesis, which in turn is fed by the vascular endothelial growth factor (VEGF) from theextracellular matrix enhanced by matrix metalloproteinase (MMP)-9. NETs themselves can recruit other neutrophils amplifying localinflammatory reaction. (C) NETs are known to trigger tumour-associated thrombosis. Indeed, cancer stimulates the release of NETs via theproduction of granulocyte colony-stimulating factor (G-CSF) or the so-called tumour microparticles with tissue factor (TF) bound on theirsurface. Since NETs are prothrombotic, their increased production fuels this vicious circle along with the interaction with von Willebrand factoranchored to the vessel wall. Besides, NET-bound NE can inactivate TF pathway inhibitor, ultimately leading to TF-dependent coagulation.(D) NETs have been described to have a role in haematogenous metastasis. NET formation within capillaries may provide a scaffold formetastasizing tumour cells, although early adhesive events represent only a part of known mechanisms.



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activation threshold of neutrophils allows a ready availabilityof NETs, but this limits the development of strong, easy andcheap diagnostic assay.209 The lack of validation of NET assaymethods might then explain the contrasting results observedso far in clinical studies. So far, (1) analytical assays (eitherfluorimetry or enzyme-linked immunosorbent assay [ELISA])for NET products, (2) confocal microscopy of neutrophilenzymes and extracellular DNA networks and (3) flow cyto-metry based on nuclear morphology or citrullinated histonesand DNA are themain approaches used for NET detection and(semi)-quantification.209 Furthermore, a standardization ofmethods and thresholds identifying an increased NET forma-tion is needed. ELISA of plasma samples is likely the methodmeeting robustness, reproducibility, and easiness criteria forNET quantification. However, ELISA is not yet able to discri-minate between increased generation and defective clearanceof NETs. Furthermore, caution should be paid to avoid post-sampling NET generation, as many physical and chemicalstimuli may impact on neutrophil activation.210

On the contrary, many concerns exist on considering NETsas a selective therapeutic target. This is largely due to thecritical role of NETs as first-line response to infectious agents.Nonetheless, in experimental PAD4�/� mouse, blocking NETo-sis is indicated as a relatively safe approach, characterized bynormal survive in septic shock andbetter outcomeafter severesterile inflammation.26 According to experimental data, manyconventional therapies used for inflammatory diseasesrevealed inhibitory properties on NET cascade. Reduced NETgeneration may occur during treatment with ROS scavengers,such as N-acetyl cysteine.211 Two small studies reported aclinical benefit of treatment with N-acetyl cysteine in patientswith SLE.212,213 Similarly, other conventional treatment usedfor the treatment of SLE, such as vitamin D and chloroquine,have been found to prevent NET generation.214,215 Alongsideknown agents, NET cascademight be inhibited bymonoclonalantibodies targeting B cells (rituximab),216,217 complementsystem (eculizumab)218–220 and potentially IFN-α (sifalimu-mab and rontalizumab).221 More recently, Cl-amidine, a PADinhibitor, has been tested in mice. The suppression of NETgeneration induced by Cl-amidine was associated withimprovementof immunecomplexdepositionandorgan injuryinmousemodels of SLE andRA.222–224Similarly, the inhibitoryeffect of Cl-amidine on NET formation has been demonstratedto reduce atherosclerosis burden, arterial thrombosis andischaemia/reperfusion injury in mice.84,225

Conclusion

As depicted in the present narrative review, a growing body ofevidence is accumulating on NETs. Although initially investi-gated in infectious diseases, research studies have expandedtheir activity in other settings, such as CV diseases, autoimmu-nityandcancer. AvailablefindingsonNETs let us think that theymay become newbiomarkers of disease activity, prognosis andpotentially therapeutic targets. Although a shroud of mysterystill surrounds NETs, we now have few answers to some topicquestions concerning NET behaviour and a wide overview ontheir significant roles.However, stillmanyefforts are requested

to clarify different aspects of NETs in both infectious and non-infectious diseases, as the mighty neutrophil surprises everyday for its unique features and protean biology.

Authors’ ContributionsA.B. and L.L. equally contributed to this work as firstauthors. A.B. and L.L. conceived the review and designedits structure. A.B., L.L., F.C., A.V. and C.D-C. collected dataand prepared the manuscript. F.M., F.D. and G.G.C. gavesuggestions to enhance the manuscript. All authorsapproved the final version of the manuscript.

Disclosure of interestAll authors declare that they have no competing interest.

AcknowledgementsThis study has been supported by a grant from theEuropean Commission (FP7-INNOVATION I HEALTH-F2–2013–602114; Athero-B cell: Targeting and exploiting Bcell function for treatment in cardiovascular disease) anda grant from the Swiss National Science Foundation toDr. F. Montecucco (no. 310030_152639/1).

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